Chromophore compositions and methods of making and using the same

ABSTRACT

Chromophore compositions and methods of making and using the same are provided. Aspects of the chromophore compositions include a chromophore component having a chromophore, such as a fluorescent dye moiety, stably associated with a prosthetic group binding cavity of a metalloprotein. Also provided are methods of making, methods of use, systems and kits related to the subject fluorescent compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 61/763,097filed Feb. 11, 2013, the disclosure of which application is hereinincorporated by reference.

INTRODUCTION

Fluorescent labeling reagents have become increasingly usefulinvestigative tools. The wider use of fluorescently labeled probes hasresulted partly from advances in instrumentation and partly from theavailability of new and improved fluorescent dyes. The squaraine dyeshave received particular interest since they are a class ofenvironmentally sensitive organic dyes showing intense fluorescence,typically in the red and near infrared region (absorption maxima arefound between 630 and 670 nm and their emission maxima are between650-700 nm). They are characterized by their unique aromatic fourmembered ring system, and are derived from squaric acid.

SUMMARY

Chromophore compositions and methods of making and using the same areprovided. Aspects of the compositions include a chromophore componenthaving a chromophore, such as a fluorescent dye moiety, stablyassociated with a prosthetic group binding cavity of a metalloprotein.Also provided are methods of making, methods of use, systems and kitsrelated to the subject fluorescent compositions.

In certain aspects, a method for encapsulating a dye in the apo form ofa heme-protein comprises providing an apo form a heme-protein comprisinga heme binding site, contacting a fluorescent dye to the protein whereinthe dye has an affinity for the heme binding site, and then reacting thedye-protein complex with a crosslinking reagent to encapsulate or lockthe dye in the protein. In some embodiments, the protein may begenetically modified to increase the affinity of the dye for the hemebinding site relative to an affinity of a heme moiety for the same hemebinding site and/or to facilitate the formation of internal crosslinks.In some embodiments the modification comprises replacing at least ahistidine amino acid with an amino acid selected from the groupcomprising alanine, leucine, phenylalanine and tryptophan. In someembodiments the method may further comprise reacting the protein with acrosslinking reagent to generate an internal crosslink in theheme-protein after the dye is bound at the heme binding site. Thecrosslinking reagent may be bis(sulfosuccinimidyl) BSOCOES(Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), DMA (Dimethyladipimidate.2HCl, DMP (Dimethyl pimelimidate.2HCl), DMS (DimethylSuberimidate.2HCl), DSG (Disuccinimidyl glutarate), DSP(Dithiobis[succinimidyl propionate]), DSS (Disuccinimidyl suberate), DST(Disuccinimidyl tartarate), DTBP (Dimethyl3,3″-dithiobispropionimidate.2HCl), or DTSSP(3,3″-Dithiobis[sulfosuccinimidylpropionate]).

Internal crosslinks may be formed by genetically modifying theheme-protein to replace any non-cysteine amino acid in the heme-proteinwith a cysteine amino acid and then reacting the genetically modifiedheme-protein with a crosslinking reagent, such as a reagent from thebismalamide family. In some embodiments the crosslinking reagent may bebis(malemido)ethane (BMOE), 1,4-bis(maleimido)butane (BMB) orbis(maleimido)hexane (BMH). In certain aspects, the heme-protein may bemyoglobin or hemoglobin. In some embodiments the fluorescent dye may hasa first quantum yield in an organic solvent that is at least 50 timeshigher than a second quantum yield in an aqueous solvent. In someembodiments the dye may be selected from Nile Red®,2-[6-[4-(dimethylamino)phenyl]-1,3,5-hexatrienyl]-3-ethyl-benzothiazoliumperchlorate (LDS 820),(2-(6-(p-dimethylaminophenyl)-2,4-neopentylene-1,3,5-hexatrienyl)-3-ethylbenzothiazoliumperchlorate) (LDS 821), coumarins, fluoranthene, mono-squaraine andbis-squaraine dye.

In certain aspects, a method of labeling an antigen in a sample such asa biological or diagnostic sample may comprise encapsulating afluorescent dye in the apo form of a heme-protein, covalently linkingthe heme-protein to an antigen specific antibody, contacting theantibody to the antigen and measuring the fluorescence of thefluorescent dye bound to the antigen. The antigen may be a cell surfacespecific marker or any antigen. The antigen may be bound to a solidsupport such as micro-titer plates or bead surfaces. The biologicalsample may comprise peripheral blood cells, urine, or saliva. In certainaspects, the heme-protein may be an apo-myoglobin or an apo-hemoglobin.

Aspects of the invention provide a composition for a fluorescencedetector (e.g., flow cytometer) measurement of an antigen in a samplecomprising a heme-protein conjugated to a reagent-capture particle ormolecule (also referred to herein as a specific binding domain) whereinthe heme-protein comprises an encapsulated fluorescent dye. In someembodiments the reagent-capture particle is an antigen specific antibodyor a solid particle having an antibody-capture reagent bound to thesurface of the particle. In certain aspects the fluorescent dyecomprises a squaraine. In certain aspects, the heme-protein may be anapo-myoglobin or an apo-hemoglobin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structures of holo-myoglobin and apo-myoglobin.

FIG. 2 provides a graph of the absorption spectrum of purifiedapo-myoglobin as compared to the absorption spectrum of holo-myoglobin.

FIG. 3A provides an exemplary chemical structure of a bis-squaraine.FIG. 3B provides an exemplary chemical structure of a mono-squaraine.

FIG. 4A provides a graph demonstrating the fluorescence of bis-squarainein the presence and absence of apo-myoglobin. FIG. 4B provides a graphdemonstrating the fluorescence of mono-squaraine in the presence andabsence of apo-myoglobin.

FIGS. 5A and 5B show graphs demonstrating the binding curves ofbis-squaraine (A) and mono-squaraine (B) to apo-myoglobin as measured bythe increased fluorescence of the dyes upon binding to the protein.

FIG. 6 shows schematic diagrams of the following: 1) binding of a dye tothe heme binding site of a heme-protein; 2) displacement of the dye by ahemin molecule; 3) internal crosslinking of the dye bound protein; and4) resistance of dye displacement by hemin when the protein encapsulateis internally cross-linked.

FIG. 7 shows titration plots of hemin titrating out mono-squaraineencapsulated in apo-myoglobin in the absence and presence of acrosslinking reagent.

FIG. 8 provides the amino acid sequence of a modified humanapo-myoglobin, where modified amino acid residues are in bold.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

The term ‘chromophore’ as used herein refers to a compound capable ofbeing detected colorimetrically or fluorometrically. The specificexamples disclosed herein describe chromophores detected by fluorescence(e.g., fluorescent dye motifs). It should be understood, however, thatthe compounds and methods described herein can equally be utilized withchromophores that are detected by other means, such as, for example,absorbance or phosphorescence. Chromophores contemplated for use in thepractice of certain embodiments of the present invention areenvironmentally sensitive fluorescent dyes wherein the quantum yield ofthe dye is significantly greater in an organic environment relative tothe quantum yield of the dye in an aqueous environment. In certainembodiments of the invention, a chromophore may be an environmentallysensitive fluorescent dye, such as a squaraine dye.

The terms “heme prosthetic group”, “heme” and “hemin” are usedinterchangeably herein and may refer to any metal bound compound of aporphyrin chelate.

The term ‘heme-protein’ refers to a metalloprotein capable of binding ahemin or heme moiety. Heme-proteins are a class of metalloproteinscontaining a hemin prosthetic group. Heme may bind to the heme-protein,either covalently or non-covalently. By ‘apo-heme-protein’ it is meant aheme-protein that does not contain a prosthetic group (e.g., hemin). By‘holo-protein’ it is meant a heme-protein that does contain a prostheticgroup (e.g., hemin). Heme-proteins contemplated for the use in thisinvention are the apo form of proteins that naturally containnon-covalently bound heme or hemin. Aspects of the invention include theuse of any heme-protein capable of non-covalently binding a heme group.

The term ‘myoglobin’ (abbreviated Mb) refers to an oxygen-bindingprotein found in the muscle tissue of vertebrates in general and inalmost all mammals. It is related to hemoglobin, which is theoxygen-binding protein in blood, specifically in red blood cells.Myoglobin is a single-chain globular protein of 153 or 154 amino acidlong wild-type sequence. Wild type human myoglobin has coordinatinghistadines (H64 and H93, as seen in FIG. 1). Myoglobin is capable ofbinding a heme (iron-containing porphyrin) prosthetic group in a cavityaround which the remaining apoprotein folds (Holo-Mb). Apo-myoglobin ismyoglobin that lacks its heme group (Apo-Mb).

The term ‘squaraine’ refers to a fluorescent dye characterized by anaromatic four membered ring system. Squaraine is derived from squaricacid, and may have one substitution on the four-membered aromatic ring(mono-squaraine), or two substitutions on the four-membered aromaticring (bis-squaraine).

DETAILED DESCRIPTION

Chromophore compositions and methods of making and using the same areprovided. Aspects of the chromophore compositions include a chromophorecomponent having a chromophore, such as a fluorescent dye moiety, stablyassociated with a prosthetic group binding cavity of a metalloprotein.Also provided are methods of making, methods of use, systems and kitsrelated to the subject fluorescent compositions.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques from molecular biology (includingrecombinant techniques), cell biology, immunoassay technology,microscopy, image analysis, and analytical chemistry, which are withinthe skill of the art. Such conventional techniques include, but are notlimited to, detection of fluorescent signals, image analysis, selectionof illumination sources and optical signal detection components,labeling of biological cells, and the like. Such conventional techniquesand descriptions can be found in standard laboratory manuals such asGenome Analysis: A Laboratory Manual Series (Vols. I-IV), UsingAntibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer:A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (allfrom Cold Spring Harbor Laboratory Press); Murphy, Fundamentals of LightMicroscopy and Electronic Imaging (Wiley-Liss, 2001); Shapiro, PracticalFlow Cytometry, Fourth Edition (Wiley-Liss, 2003); Herman et al,Fluorescence Microscopy, 2nd Edition (Springer, 1998); all of which areherein incorporated in their entirety by reference for all purposes.

In further describing embodiments of the invention, aspects ofembodiments of the compositions and methods for their fabrication willbe described first in greater detail. Next, embodiments of methods ofusing the compositions, as well as systems and kits that may be used inpracticing methods of the invention, are reviewed in greater detail.

Compositions

As summarized above, compositions of the invention are chromophorecompositions that include a chromophore stably associated with aprosthetic group binding cavity of a metalloprotein. Each of thesecomponents is now described in greater detail.

Metalloprotein Component

As summarized above, the compositions include a metalloprotein having aprosthetic group binding cavity. The term ‘metalloprotein’ refers to aprotein having a metal ion in its structure, e.g., as provided by aprosthetic group. Metalloproteins of interest are proteins having aprosthetic group binding cavity. A wild-type metalloprotein is capableof binding a prosthetic group having one or more metal ions. Theprosthetic group binding cavity may, in a wild type metalloprotein, becapable of associating with (e.g., binding to, encapsulating, foldingaround, etc.) a prosthetic group.

Depending on the nature of the metalloprotein, the prosthetic groupbinding cavity may vary greatly. Prosthetic groups for which the bindingcavity of the matalloprotein of interest (or at least in the wild typeversion thereof) include, but are not limited to macrocyclic ligands,such as ligands having a ring of 8 or more, 9 or more, or 10 or moreatoms. For example, the prosthetic group may be a heterocyclic organicring, such as porphyrin. When present as part of the metalloprotein, theprosthetic group may include one or more metal ions (e.g., complexed toatoms of a heterocyclic organic ring). In certain embodiments, theprosthetic group may be a heme compound.

By prosthetic group binding cavity is meant a hollow space, e.g., pit orindentation, in the three dimensional structure of the metalloproteinthat, at least in the wild type version of the metalloprotein ofinterest, is configured to at least partially house or encompass aprosthetic group to thereby stably associate with the prosthetic group.The volume of the prosthetic group binding cavity of the metalloproteinmay vary, ranging in some instances from 50 nm³ to 1,000 nm³, such as2500 nm³ to 1000 nm³, an including 500 nm³ to 1,000 nm³, wherein in someinstances the volume of the binding cavity is 100 nm³ or more, such as250 nm³ or more, and including 500 nm³ or more, and in some instancesthe volume is 1,000 nm³ or less, such as 1750 nm³ or less. Theprosthetic group may associate with the prosthetic group binding cavityof a wild type metalloprotein by any of a number of non-covalentinteractions, such as non-polar, polar, and ionic interactions, whichprovide for association of the prosthetic group with the metalloprotein.As indicated above, the prosthetic group binding cavity may be capableof encompassing all or part of the prosthetic group.

Metalloproteins of interest include animal metalloproteins, e.g.,vertebrate metalloproteins, including fish, amphibian, reptile, bird anda mammalian metalloproteins. In some instances, the metalloprotein is amammalian metalloprotein. The terms “mammal” or “mammalian,” where areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), ungulates (e.g., horses, cows, pigs),whales, and primates (e.g., humans, chimpanzees, and monkeys).

The metalloprotein of the compositions described herein may be awild-type protein or a homologue or mutant thereof. Homologs or proteins(or fragments thereof) that vary in sequence from wild type amino acidsequences metalloproteins as described herein are found in compositionsof the invention in certain embodiments. By homolog is meant a proteinhaving 10% or more, such as 20% or more and including 30% or more, andin some instances 35% or more, such as 40% or more and including 60% ormore amino acid sequence identity to a wild type metalloprotein ofinterest (such as a wild type heme-protein, e.g., a wild typeapomyoglobin, such as a wild type mammalian apomyoglobin, e.g., humanapomyoglobin), e.g., as determined using MegAlign, DNAstar (1998)clustal algorithm as described in D. G. Higgins and P. M. Sharp, “Fastand Sensitive multiple Sequence Alignments on a Microcomputer,” (1989)CABIOS, 5: 151-153. (Parameters used are ktuple 1, gap penalty 3,window, 5 and diagonals saved 5). In some embodiments, homologues ofinterest have much higher sequence identify to a corresponding wild typeprotein, e.g., 65%, 70%, 75%, 80%, 85%, 90% or higher. Also provided areproteins that are substantially identical to the wild type protein,where by substantially identical is meant that the protein has an aminoacid sequence identity to the sequence of wild type protein of 60% ormore, such as 65% or more and including 70% or more, where in someinstances the identity may be much higher, e.g., 75%, 80%, 85%, 90%, 95%or higher.

Proteins which are mutants of the naturally occurring proteins are alsopresent in compositions of invention according to certain embodiments.Mutants include single amino acid changes, deletions of one or moreamino acids, insertions of one or more amino acids, N-terminaltruncations, C-terminal truncations, etc. Mutants can be generated usingstandard techniques of molecular biology, e.g., random mutagenesis, andtargeted mutagenesis.

In certain aspects, the metalloprotein of the subject compositions is aheme-binding protein (or “heme-protein”) that binds to a heme prostheticgroup, or mutant of a such a heme-binding protein. An ‘apo’ heme-proteinis a heme-protein that does not contain a prosthetic group (e.g.,hemin). A ‘holo’ heme-protein is a heme-protein that does contain aprosthetic group (e.g., hemin). Heme-proteins provided in theembodiments herein may be the apo form of proteins that would naturallycontain non-covalently bound heme or hemin, or mutants of such proteins,as reviewed in greater detail below. In some embodiments, theheme-binding protein may be a hemoglobin or a myoglobin. In someembodiments the heme-protein may be apomyoglobin (Apo-Mb) orapohemoglobin (Apo-Hb). The heme-protein may have a naturally occurring(wild type) amino acid sequence. For example, the heme-protein may havethe sequence of a naturally occurring mammalian apomyoglobin, such ashuman, mouse, horse, whale, or pig apomyoglobin. The genbank accessionnumbers of apomyoglobin protein sequences of interest include, but arenot limited to: NP_(—)976312.1, NP_(—)976311.1, NP_(—)005359.1,CAA25109.1, AAH14547.1, NP_(—)005359.1, NP_(—)067599.1,NP_(—)001157520.1, NP_(—)038621.2, NP_(—)001157488.1, NP_(—)999401.1,AAA31073.1, NP_(—)776306.1, NP_(—)001161224.1, NP_(—)001072126.1,ABN71515.1, NP_(—)001274714.1, NP_(—)001273527.1, ADQ74520.1,ADQ74518.1, ADQ74517.1, ADQ74514.1, ADQ74513.1, ADQ74511.1, ADQ74510.1,ADQ74507.1, CAA27994.1, NP_(—)956880.1, AAH56727.1, AAR00323.1, 101 M_A,NP_(—)001125556.1, NP_(—)001165333.1, NP_(—)001187526.1,NP_(—)001266612.1, NP_(—)001273527.1, NP_(—)001274714.1, AGM75765.1,AGM75763.1, AGM75762.1, AGM75761.1, AGM75760.1, AGM75759.1, AGM75770.1,XP_(—)001081975.2, XP_(—)001156646.1, BAF03579.1, and the like.

As reviewed above, in some instances the metalloprotein component of thecompositions may be a mutant metalloprotein. As such, also of interestare mutants of wild type heme-proteins, e.g., as described above.Metalloproteins found in some instances of the compositions are mutantsof mammalian apomyoglobins, i.e., apomyoglobin proteins that include oneor more mutations, such as described above, as compared to a wild-typeprotein. The mutations may vary, where mutations of interest includesubstitution, deletion, insertion, and point mutations which result in amodified amino acid sequence, as compared to a reference wild typesequence.

In certain aspects, a mutation may be a point mutation. In someembodiments, the point mutation may include (result in) a substitutionof a cysteine residue for a naturally occurring amino acid residue. Thesubstituted cysteine residue may be proximal to or within the prostheticgroup binding cavity of the metalloprotein. In some embodiments, a pointmutation may include a substitution of a non-histidine residue for anaturally occurring histidine residue. For example, the non-histidineresidues may be selected from the group including alanine, leucine,phenylalanine, tryptophan. In some embodiments, the mutation may includea substitution of a non-histidine residue for a heme coordinatinghistidine residue, such as H64 and H93 of human myoglobin, e.g., as seenin FIG. 1. In this example, the mutation may improve an association ofthe fluorescent dye moiety with the prosthetic binding group cavity(heme binding site). The mutation may, alternatively or in addition,decrease an affinity of the heme binding site for heme.

In certain aspects, the metalloprotein may be an apomyoglobin having amutation altering the protein structure, cavity size, affinity of theheme-binding site (i.e. cavity) to heme, affinity of the heme-bindingsite to a fluorescent dye moiety, having a mutation that enhance aninternal crosslinking upon exposure of the myoglobin to a crosslinkingreagent, or any combination thereof. For example, the apomyoglobin maybe a human myoglobin or a myoglobin of another animal, wherein one ormore amino acid residues are replaced at any of the following positions(starting after the initiator methionine): Trp 14, Lys 16, His 24, Gln26, Val 28 (or Ile 28 in certain non-human species), Leu 29, Ile 30, Phe33, Lys 34, His 36, Phe 43, Asp 44, Lys 45 (or Arg 45 in certainnon-human species), Phe 46, Lys 56, Asp 60, Leu 61, His 64, Thr 67, Val68, Leu 69, Ala 71, Leu 89, Gln 91, Ile 99, Leu 104, Ile 107 (or Leu 107in certain non-human species), Ser 108, His 119, Leu 137, and Phe 138.In certain aspects, a non-histidine amino acid, such as alanine,leucine, phenylalanine or tryptophan, may replace a histidine, such asHis 24, His 36, His 64, and His 119 of the above example. In certainaspects, one or more of the amino acid residues Val 28 (or Ile 28 incertain non-human species), Val 68, Ile 107, Leu 89, Leu 104, and Phe138 may be replaced with another amino acid, such as tryptophan. Incertain aspects, a substitution of an amino acid of the apomyoglobin,such as any listed in the above, may enhance an internal crosslinkingupon exposure of the apomyoglobin to a crosslinking reagent. Forexample, a cysteine residue may be substituted for any of the previouslylisted amino acids. Alternatively or in addition, a lysine residue maybe substituted for any of the above-listed amino acids. Discussion ofmutations of myoglobin that may be suitable to embodiments describedherein may be found in, among other locations, “Discovery of new ligandbinding pathways in myoglobin by random mutagenesis” (Huang et al.Nature Structural Biology 1994; 1, 226-229.

In certain aspects, the metalloprotein (e.g., the heme-binding protein)may have a tag, such as a His tag, CBP, MBP, GST, or other tag foraffinity purification.

An example of a mutant human apomyoglobin of interest in certainembodiments is a protein having the sequence shown in FIG. 8 (i.e., SEQID NO:01). As illustrated in FIG. 8, amino acids 3 to 14 are of ahistidine tag enabling purification by Ni-NTA affinity. M27 of theillustrated sequence corresponds to the initiator methionine of thewild-type human myoglobin sequence. Amino acids 28 to 180 correspond toa human myoglobin sequence of 153 amino acids with the followingmutations: H64 of the wild-type sequence has been replaced with alanine(A91 of SEQ ID NO:01); H93 of the wild-type sequence has been replacedwith phenylalanine (F120 of SEQ ID NO:01); and amino acid residues aboveand below the edge of the heme-binding site were replaced with cysteines(C94 and C119 of SEQ ID NO:01). In certain aspects, the metalloproteinmay include the amino acid sequence starting with M27 or G28 of SEQ IDNO:01.

Chromophore Component

As summarized above, compositions as described herein further include achromophore. As reviewed above, the chromophore may be any compoundcapable of being detected colorimetrically or fluorometrically. Thespecific examples disclosed herein describe chromophores detected byfluorescence. It should be understood, however, that the compounds andmethods described can equally be utilized with chromophores that aredetected by other means readily available to those skilled in the art,such as, for example, absorbance or phosphorescence.

Chromophores of interest include fluorescent dye moieties. Thefluorescent dye moiety may be any suitable molecule that fluoresces whenassociated with the metalloprotein. In certain aspects, the fluorescentdye moiety is a non-protein organic fluorophore. The molecular weight ofthe non-protein organic fluorophore may vary, ranging in some instancesfrom 50 Da to 5 kDa, such as 100 Da to 1 kDa, including 200 Da to 500Da.

In certain embodiments, a non-protein organic fluorophore may include amolecule belonging to any of the following chemical families: xanthenederivatives (such as fluorescein, rhodamine, Oregon green, eosin, Texasred, etc.); fluorescein derivatives (such as 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluoresceinisothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein,and QFITC(XRITC)); acridine derivatives (such as acridine orange,acridine yellow, acridine red, acridine isothiocyanate, proflavin,etc.); quinone-imine derivatives (such as azines, oxazines, andthiazines); cyanine derivatives (such as cyanine, indocarbocyanine,oxacarbocyanine, thiacarbocyanine, merocyanine, etc.); naphthalenederivatives (such as dansyl and prodan derivatives); coumarin andderivatives thereof (e.g., 7-amino-4-methylcoumarin (i.e., AMC, Coumarin120), 7-amino-4-trifluoromethylcouluarin (i.e., coumaran 151)); azoderivatives; oxadiazole derivatives (such as pyridyloxazole,nitrobenzoxadiazole benzoxadiazole, etc.); anthracene derivatives;anthraquinones (such as DRAQ5, DRAQ7 CyTRAK Orange, etc.); pyrenederivatives (such as pyrene butyrate, succinimidyl 1-pyrene butyrate,cascade blue, etc.); oxazine derivatives (such as Nile red, Nile blue,cresyl violet, oxazine 170, etc.); arylmethane derivatives (such asauramine, crystal violet, malachite green, etc.); tetrapyrrolederivatives (such as porphin, phthalocyanine, bilirubin, etc.);squaraines (e.g., bis-squaring, mono-squaraine), squarylium,2-[6-[4-(dimethylamino)phenyl]-1,3,5-hexatrienyl]-3-ethyl-benzothiazoliumperchlorate (LDS 820),(2-(6-(p-dimethylaminophenyl)-2,4-neopentylene-1,3,5-hexatrienyl)-3-ethylbenzothiazoliumperchlorate) (LDS 821), fluoranthene,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS; anthranilamide, 5′,5″-dibromopyrogallol-sulfonephthalein(Bromopyrogallol Red); diethylenetriamine pentaacetate,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,[4-dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), erythrosinand derivatives thereof (such as erythrosin B and erythrosinisothiocyanate), fluorescamine and derivatives thereof, Malachite Greenisothiocyanate, 4-methylumbelliferone, ortho cresolphthalein,nitrotyrosine, pararosaniline, Phenol Red, B-phycoerythrin,o-phthaldialdehyde, Reactive Red 4 (Cibacron™ Brilliant Red 3B-A),rosolic acid and terbium chelate derivatives, and so forth. A number ofdyes are described in “Fluorescent Dyes and Their SupramolecularHost/Guest Complexes with Macrocycles in Aqueous Solution” (Dsouza etal. Chem. Rev. 2011, 111, 7941-7980), where such dyes may be present incompositions described herein. In addition, the non-protein organicfluorophore may include one or more macrocyclic ligands.

In certain aspects, the fluorescent dye moiety (e.g., non-proteinorganic fluorophore) may be an environmentally sensitive fluorophore.For example, the fluorescent dye moiety may be insoluble in an aqueousenvironment or may demonstrate low solubility in an aqueous environment.The environmentally sensitive fluorophore may exhibit at least one of afluorescence excitation spectrum, a fluorescence emission spectrum, afluorescence intensity (e.g., quantum yield), and a fluorescencelifetime that differs depending on the environment (e.g., an aqueous vs.an organic environment, the pH of the environment, etc.) in which it ispresent. For example, an environmentally sensitive fluorophore may havea fluorescence intensity (e.g., a quantum yield for a given excitationwavelength, intensity of a fluorescence emission maxima, etc.) that ishigher when the fluorophore is in an organic as compared to an aqueousenvironment. In certain embodiments, an environmentally sensitivefluorophore stably associated with the prosthetic group binding cavityof the metalloprotein as found in compositions of the invention may havea first fluorescence intensity that is higher (e.g., 50 times higher ormore) than a second fluorescence intensity of the environmentallysensitive fluorophore free in aqueous solution. For example, the firstfluorescence intensity may be at least 5 times higher, at least 10 timeshigher, at least 20 times higher, at least 50 times higher, at least 100times higher, or at least 200 times higher than the second fluorescenceintensity. An example of such a fluorophore (i.e., fluorescent dyemoiety) is illustrated in FIG. 4, where FIG. 4A and FIG. 4B show thefluorescence of bis-squaraine (A) and mono-squaraine (B) in an aqueoussolution in the presence and absence of apomyoglobin. It can be seenthat upon binding to apomyoglobin, the squaraine dyes show a greaterthan 50 fold increase in fluorescence in the 630 to 700 nm range.

In certain aspects, a chromophore that binds to a heme-protein may beutilized in the subject compositions. The chromophore may be anenvironmentally sensitive fluorescent molecule wherein the quantum yieldof the molecule is greater (e.g., greater than 5, 10, 50, or 1000 times)in an organic environment relative to the quantum yield of the moleculein an aqueous environment.

In certain embodiments, an environmentally sensitive fluorophore may bea derivative of squaraine, dapoxyl, coumarin, cucurmin, badan, DMABN,HMPO, ThT (thioflavin T), bisimides (e.g., DBN), styrlpyridinium (e.g.,2-ASP, 4-ASP), benzimidazole (e.g., BEA, MBC, bis-benzimidazole),2-styrylindolium dye (e.g., STIND1, STIND2), ADMP, DAPS, cucurbiturils,adamantlyl, naphthalimides, 1, 8-1-anilinonaphthalene-8-sulfonic acid(1,8-ANS), 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS),2-(p-toluidinyl), naphthalene-6-sulfonic acid (2,6-TNS),6-propionyl-2-dimethylaminonaphthalene (PRODAN), or any other suitableenvironmentally sensitive fluorophore, such as those described by Dsouzaet al. (Chem. Rev. 2011, 111, 7941-7980).

In certain aspects, the environmentally sensitive fluorophore may be asquaraine, characterized by a four-membered aromatic ring. Squaraine maybe derived from squaric acid, and may have one substitution(mono-squaraine) or two substitutions (bis-squaraine). A Bis-squarainemay be either symmetric or asymmetric. Examples of chemical structuresof a bis-squaraine and a mono-squaraine are presented in FIGS. 3A and 3Brespectively. Any suitable squaraine may be used in the subjectembodiments, including those described by “β-Cyclodextrin as aphotosensitizer carrier: Effect on photophysical properties and chemicalreactivity of squaraine dyes” (K. T. Arun et al. J. Phys. Chem. B. 2011,115, 7122-7128), described by Dsouza et al. (Chem. Rev. 2011, 111,7941-7980), and provided by suppliers such as Sigma-Aldrich and otherchemical suppliers.

Metalloprotein-Chromophore Component

As summarized above, compositions of the invention include achromophore, such as a fluorescent dye moiety, stably associated with aprosthetic binding cavity of a metalloprotein or mutant thereof. Bystably associated is meant that the chromophore component is notdisplaced from the metalloprotein, at least under conditions of intendeduse, e.g., when employed in methods as described in greater detailbelow.

The fluorescent dye moiety may be non-covalently bound to the prostheticgroup binding cavity (such as by non-polar interactions, polarinteractions, ionic interactions, steric hindrance, etc.). In certainaspects, the fluorescent dye moiety may be stably associated with theprosthetic group binding cavity of the metalloprotein. A stableassociation between the fluorescent dye moiety and the metalloproteinmay be characterized by a K_(d) of 1 mM or less, 100 μM or less, 10 μMor less, 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 100μM or less, 10 μM or less, and so forth. FIG. 5A and FIG. 5B demonstratethat the binding constants of the environmentally sensitive dyes to theheme proteins may be measured by monitoring the increased fluorescenceof the dye upon binding to the apo-heme protein. Molecular modelingstudies and the experimentally determined Kd for bis-squaraine, andmono-squaraine (2-20 nM) demonstrate a strong affinity of thefluorescent molecules for the heme binding site of apo-Mb.

In certain aspects, the association of the fluorescent dye moiety withthe cavity may be enhanced by a stabilizing modification. Stabilizingmodifications are modifications of the metalloprotein that are made toenhance the association of the chromophore with the metalloprotein, andincrease the stability of the association of these two components witheach other relative to a control, at least to a measurable extent. Thestabilizing modification may vary, and in some instances may be aninternal crosslinking of amino acid residues of the metalloprotein.Internal crosslinkings of interest are those which serve to secure theassociation of the dye moiety in the cavity, and thereby enhance thestable associate of dye moiety with the metalloprotein. Any convenientresidues may be joined to each other to provide the desiredcrosslinking. Residues of interest include those which are proximal tothe prosthetic group binding cavity and are amenable to crosslinking. Inone aspect, the internal crosslinking may include crosslinked cysteineresidues. The internal crosslinking may alternatively or additionallyinclude crosslinked lysine residues. A list of crosslinking reagents andprotocols is described in, among other locations, the website made byplacing “https://www.” before“biochem.wisc.edu/faculty/weibel/lab/methods/Crosslinking_Reagents_Pierce.pdf”.Specific crosslinking reagents of interest include, but are not limitedto: bis(sulfosuccinimidyl) BSOCOES(Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), DMA(Dimethyladipimidate.2HCl, DMP (Dimethyl pimelimidate.2HCl), DMS (DimethylSuberimidate.2HCl), DSG (Disuccinimidyl glutarate), DSP(Dithiobis[succinimidyl propionate]), DSS (Disuccinimidyl suberate), DST(Disuccinimidyl tartarate), DTBP (Dimethyl3,3″-dithiobispropionimidate.2HCl), or DTSSP(3,3″-Dithiobis[sulfosuccinimidyl-propionate]). The internalcrosslinking may ensure that the fluorescent dye moiety remains bound tothe cavity (e.g., the heme-binding site of a heme protein). Aspects ofthis invention include any number of crosslinked amino acids (e.g., one,two, three or more crosslinks) in the metalloprotein that act tonon-covalently lock the protein to the docked fluorescent molecule, asseen in FIG. 6 and FIG. 7.

A given chromophore composition of the invention may include a singlechromophore/metalloprotein component (such as described above) or two ormore such components stably associated, e.g., covalently ornon-covalently bound, to each other, such as two or more, three or more,five or more, ten or more, including twenty or more such componentsstably associated with the each other. Where a given compositionincludes two or more such components, the components may be the same ordifferent from each other, e.g., differing from each other with respectto the nature of the chromophores and/or metalloproteins.

Specific Binding Domain

In certain aspects of the invention, the composition may include one ormore specific binding domains. The specific binding domain may beassociated with the metalloprotein in a variety of difference ways,e.g., it may be conjugated by a linker, fused, or otherwise covalentlybound to the composition, e.g., to a metalloprotein of the composition.In some aspects, the specific binding domain may have an affinity for ananalyte of K_(a) of 10⁴ M⁻¹ or greater, 10⁵ M⁻¹ or greater, 10⁶ M⁻¹ orgreater, 10⁷ M⁻¹ or greater, 10⁸ M⁻¹ or greater, 10⁹ M⁻¹ or greater,10¹⁰ M⁻¹ or greater, 10¹¹ M⁻¹ or greater, 10¹² M⁻¹ or greater. Thecomposition may include a peptidic or polypeptidic moiety. In someaspects, the peptidic or polypeptidic moiety may include an antibody ora binding fragment thereof (e.g., a monovalent antibody). In someaspects, the specific binding domain may be a ligand (e.g., growthfactors, streptavidin, cytokines, and so forth) that specifically bindsa cell surface receptor. In some aspects, the specific binding domainmay be a nucleic acid moiety (such as an aptamer).

In certain aspects of the invention, the composition may include alinker conjugated to (e.g., covalently bound to) the metalloprotein. Thelinker may be any linker molecule known in the art such as succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or others. Thelinker may be functionalized (e.g., to covalently attach to an antibodyor other specific binding domain) and may be bi-functional. In someaspects, the linker may be selected from one of any of the crosslinkerreagents discussed in embodiments of the subject compositions.

In certain aspects, the heme-protein may be covalently conjugated to anantibody or solid support. Conjugation to an antibody or solid supportmay occur via any means, such as via succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or via another linermolecule. The complexes of this invention may be conjugated to a solidsupport such as beads or columns or the like. Non-antibody proteins mayalso be labeled with the protein-dye complexes (e.g., fluorescentcomponents) disclosed herein. For example, antigens (e.g. ligands for acell surface receptor or an antibody) may be coupled via any means withthe protein-dye complexes.

Methods of Making

Aspects of the invention further include methods of making any of thesubject compositions, e.g., as described above. A method of producing achromophore composition may include contacting a metalloprotein havingan empty prosthetic group binding cavity with a fluorescent dye moietyin a manner sufficient for the fluorescent dye moiety to stablyassociate with the metalloprotein, e.g., by entering the prostheticgroup binding cavity. The method may further enhancing the associationof the dye moiety with the metalloprotein, e.g., by producing astabilizing modification of the protein, such as described above.

In certain aspects, stably associating the fluorescent dye moiety withthe prosthetic group binding cavity may include modifying themetalloprotein. Modifying the metalloprotein may include generating aninternal crosslink in the metalloprotein after the dye enters theprosthetic group binding cavity, such as through the use of any of thecrosslinker reagents discussed in embodiments of the subjectcompositions. In certain aspects, a cysteine residue of themetalloprotein may replace a non-cysteine residue of a naturallyoccurring metalloprotein. Generating the internal crosslink in themetalloprotein may include crosslinking a cysteine residue that replacesa non-cysteine residue of a naturally occurring metalloprotein. Use ofalternate crosslinkers to collapse the dye cavity may be employed toensure the dye is not displaced when used in complex blood solutions. Alarge family of cross-linkers may successfully connect protein/dyemonomers (fluorescent components) to establish signal amplification.

The use of internal crosslinking reagents may insure that theencapsulated fluorescent molecule remains bound even in the presence ofmolecules capable of competitively binding to the heme site (e.g.,hemin). This is schematically illustrated in FIG. 6, and supported byFIG. 7. Aspects of this invention may include any number of crosslinkedamino acids (e.g., one, two, three or more crosslinks) in the proteinthat act to non-covalently lock the protein to the docked fluorescentmolecule (e.g., at the prosthetic binding group cavity). Internalcrosslinking may be achieved in any number of ways in order to lock thedye into the prosthetic binding group cavity (e.g., the heme-bindingsite). In some embodiments crosslinking reagents such asbis(sulfosuccinimidyl) may be reacted with the native or geneticallymodified protein after the fluorescent molecule has been bound to theprotein. For example, human recombinant apo-Mb may be incubated with anexcess of mono-squaraine in a buffer such as a buffer including 50 mMsodium phosphate buffer, 150 mM sodium chloride, pH 7.2. The sample maybe incubated in the presence of a crosslinking reagent. For example,samples of the apo-Mb/mono-squaraine complex may be incubated for 2hours with an excess of BS3 reagent (such as a 2, 7, 10, 15 or more foldexcess).

As the structure of the metalloprotein may be sensitive to pH, anoptimal pH range for association of the prosthetic binding cavity of themetalloprotein with the fluorescent dye motif may exist. In certainaspects, the method may include modulating pH prior to generating aninternal crosslink in the metalloprotein. For example, the pH of thecomposition may be buffered to be anywhere from 5 to 9, such as 6 to 8,6.5 to 7.5, 5 to 5.5, 5.5 to 6, 6 to 6.5, 6.5 to 7, 7 to 7.5, 7.5 to 8,8 to 8.5, 8.5 to 9, and so forth.

The metalloprotein (e.g., such as a modified heme-protein) may bereacted with reagents from the bismaleimide family or any othercrosslinking reagent that reacts with cysteine residues in order to forminternal crosslinks in the heme protein after it is complexed with afluorescent molecule (e.g., fluorescent dye moiety). In some embodimentsthe crosslinking reagent may be bis(maleimido)ethane (BMOE),1,4-bis(maleimido)butane (BMB) or bis(maleimido)hexane (BMH). In someembodiments, the crosslinking reagent may be bis(sulfosuccinimidyl)BSOCOES (Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone) DMA (Dimethyladipimidate.2HCl, DMP (Dimethyl pimelimidate.2HCl), DMS (DimethylSuberimidate.2HCl), DSG (Disuccinimidyl glutarate), DSP(Dithiobis[succinimidyl propionate]), DSS (Disuccinimidyl suberate), DST(Disuccinimidyl tartarate), DTBP (Dimethyl3,3″-dithiobispropionimidate.2HCl), or DTSSP(3,3″-Dithiobis[sulfosuccinimidyl-propionate]). Other suitablecrosslinking reagents include imidoesters, N-hydroxysuccinimide-esters(NHS-esters), maleimides, paraformaldehyde, glutaraldehyde, haloacetyls,pyridyl disulfides, hydrazides, carbodiimides, aryl azides, isocyanates,vinyl sulfones and any other cross-linking reagents disclosed herein.Crosslinking reagents may include, but are not limited, to thosedescribed by U.S. publication number 20110177617, which is incorporatedherein by reference.

In certain aspects, the heme-protein may be covalently conjugated to anantibody or solid support before or after the binding of the fluorescentmolecule for use as a biological detection agent. Conjugation to anantibody or solid support may occur via any means such as via linkermolecules known in the art such as succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), streptavidin,avadin, or any of the crosslinking reagents described herein. Thecomplexes of this invention may be conjugated to a solid support such asbeads or columns or the like. Non-antibody proteins can also be labeledwith the protein-dye complexes disclosed herein.

Aspects of the invention further include methods of producing ametalloprotein of any of the subject compositions previously described.In certain aspects, a method of producing a metalloprotein suitable forbinding to a fluorescent dye moiety may include expressing a mutantmetalloprotein in a host cell, wherein the mutant metalloproteinincludes a mutation in a prosthetic group binding cavity. The method mayfurther include harvesting the mutant metalloprotein from the host cell.

In certain aspects, the metalloprotein may be a heme binding protein,such as an apomyoglobin having one or more mutations as previouslydescribed. In certain aspects, the host cell may be of any suitablebacterial strain, such as Escherichia coli, Corynebacterium,Staphylococcus, Pseudomonas fluorescens and so forth. Alternatively, thehost cell may an animal cell, such as a mammalian cell line or a yeaststrain (e.g., Saccharomyces cerevisiae, Pichia pastoris, etc.). The stepof expressing a mutant metalloprotein in the host cell may includeexposing the host cell to a vector including a nucleotide sequenceencoding the mutant metalloprotein. The vector may be a plasmid, virus(e.g., a bacteriophage), or any suitable vector for expressing thenucleotide sequence. The vector may include a sequence encoding anaffinity tag (such as a His tag, CBP, MBP, GST, etc.). The nucleotidesequence may be under the control of a promoter, an operator (e.g., aTet operator), an operon (such as the Lac operon), or any combinationthereof. Expression of the nucleotide sequence may be inducible orconstitutive. In certain aspects, the vector may include one or moreantibiotic resistance genes, and the step of expressing themetalloprotein may include exposing the host cells (i.e., a bacterialstrain) to an antibiotic to select for transformed bacteria. In certainaspects, the step of expressing may further include exposing the hostcell to an inducing agent (such as IPTG, Tetracycline, Doxycycline, andderivatives thereof). For example, the host cell may be a bacterialstrain and the step of expressing may include transforming the host cellwith a plasmid including the nucleotide sequence. Recombinant techniquesand protein expression systems and methods are discussed in “Recombinantprotein expression and purification: A comprehensive review of affinitytags and microbial applications” (Young et al. Biotechnol J. 2012, 7(5):620-34), and “Recombinant Gene Expression: Reviews and Protocols”(Palomares et al. Methods Mol. Biol. 2004, 267:15-52). A list of vectorsand protocols is also provided at the website produced by placing“http://www.” before“labome.com/method/Recombinant-Protein-Expression-Vector-Host-Systems.html”.

In certain aspects, the step of harvesting the metalloprotein mayinclude growing the host cell culture (e.g., to confluency in anadherent animal cell culture, to an OD such as an OD600 of 0.6 to 0.8,0.8 to 1, 1 to 1.2 for an yeast or bacterial cell culture, etc.). Thestep of harvesting may further include generating a host cell lysate byany suitable method. The step of harvesting may still further includepurifying the metalloprotein by any suitable method (e.g.,size-exclusion chromatography, ion-exchange chromatography, affinitybeads or chromatography such as Ni-NTA beads or column, and so forth).In certain aspects, the metalloprotein may include an affinity tag (suchas a His tag, CBP, MBP, GST, and so forth) that is used to purify theprotein.

The protein may be naturally occurring or modified (e.g., genetically orchemically) in any way. In some embodiments genetic modification mayinclude modification or replacement of any amino acid in order toincrease the affinity of the protein for the chromophore relative to theaffinity of the protein for hemin. For example, heme binding histidineresidues in the heme-protein may be replaced with any other amino acid,such as alanine, leucine, phenylalanine and tryptophan. In someembodiments the genetic modification may include the replacement of oneor more non-cysteine amino acid with a cysteine amino acid in order tofacilitate internal crosslinking of the protein.

In certain aspects, the method may include genetically modifying ametalloprotein nucleotide sequence to provide a sequence encoding ametalloprotein suitable for binding to a fluorescent dye moiety, priorto expressing the modified sequence in the host cell. Suitabletechniques for genetically modifying the metalloprotein nucleotidesequence include but are not limited to site directed mutagenesistechniques such as cassette mutagenesis, PCR site-directed mutagenesis,whole plasmid mutagenesis. Any suitable recombinant techniques (e.g.,PCR, restriction digestion and ligation, etc.) may be used to provide avector (e.g., virus, plasmid) having the modified nucleotide sequence.The modified nucleotide sequence may include one or more mutations thatresult in any of the metalloproteins of the subject compositionpreviously described.

In some embodiments non-histidine amino acids may be substituted forhistidine amino acids via recombinant genetic techniques. In otherembodiments cysteine amino acids may be substituted for non-cysteineamino acids via recombinant genetic techniques. Substitution of acysteine amino acid for a non-cysteine amino acid may enhance aninternal crosslinking upon exposure of the metalloprotein to a reagentfrom the bismaleimide family or any other crosslinking reagent thatreacts with cysteine residues.

Aspects of the invention may include vectors for use in practicing theabove methods.

Methods of Use

Aspects of the invention further include methods of using the subjectcompositions. A method of assaying a sample may include contacting thesample with a fluorescent composition including; a metalloproteinincluding a prosthetic group binding cavity, and a fluorescent dyemoiety stably associated with the prosthetic group binding cavity. Incertain aspects, the metalloprotein may include a stabilizingmodification that enhances association of the dye moiety with thecavity. The fluorescent composition may be any of the subjectcompositions described above.

In certain aspects, the fluorescent composition may include a specificbinding domain, according to any of the embodiments of the subjectcompositions described above. The method may further include obtaining afluorescence signal from the sample. The fluorescence signal may beprovided by the fluorescence composition (e.g., by a fluorescent dyemoiety of the composition). In certain aspects, the fluorescence signalmay be obtained by exposing the sample to monochromatic light or a widerange of light in the UV, visible, or infrared range of theelectromagnetic spectrum. The fluorescence signal may be a fluorescenceemission maxima (e.g., in the UV, visible, or infrared range). Themethod may further include evaluating an analyte bound by the specificbinding domain, based on the fluorescence signal. In certain aspects,evaluating may include assessing the amount of the analyte in thesample. The amount of the analyte may be assessed as positively relatingto the intensity of the fluorescence signal. In certain aspects, themethod may include assessing the amount of a ligand as an inversefunction of the intensity of the fluorescence signal, wherein the ligandcompetes for the analyte bound by the specific binding domain (e.g., asin the case of a competitive immunoassay).

The sample may be a biological sample, such as a cell sample. In certainaspects, the sample may include a biological fluid, such as blood (e.g.,whole blood, serum, plasma), urine, saliva, and so forth. In certainaspects, the sample may be a cell sample such as a peripheral bloodmononuclear cell sample, a tissue sample (e.g., a cross section of asolid tissue), a cell culture, and so forth. In some aspects, the samplemay include free heme such as may be present in a blood sample.

In certain aspects, the method may involve assaying the sample by flowcytometry, microscopy (e.g., of a tissue or cell culture sample stainedwith the fluorescent composition), immunoassays, immunostaining, or acombination thereof. The complexes (fluorescent compositions) of theembodiments described herein can be used to label a variety ofnon-protein molecules. Chen and Evangelista, for example, describe amultianalyte drug assay wherein labeled morphine and phencyclidine (PCP)are used in a competitive immunoassay to detect drug levels in urine(Chen and Evangelista, Clin. Chem. 40(9):1819-1822, 1994). Antibodyconjugates (e.g., fluorescent compositions including an antibodyspecific binding domain) can also be used for diagnostic purposes, bothin vitro and in vivo. Ballou et al., for example, described a method ofusing antibody conjugates for the detection of tumors in vivo. (Ballouet al., Cancer Immunol. Immunother. 41(4):257-263, 1995.) Lanza et al.describe an in vitro method of diagnosing leukemia using antibodyconjugates. (Lanza et al., Leuk. Lymphoma 18(Suppl. 1):25-30, 1995). Incertain aspects, the specific binding domain may be a ligand (e.g.,growth factors, streptavidin, cytokines, and so forth) specific for acell surface receptor, and may be used to study ligand:receptorinteractions for either research or clinical purposes.

Systems

Aspects of the invention further include systems for use in practicingthe subject methods. A sample analysis system may include a flow channelloaded with a sample having a fluorescent composition. The fluorescentcomposition may include a metalloprotein having a prosthetic groupbinding cavity and a fluorescent dye moiety stably associated with theprosthetic group binding cavity. In certain aspects, the system may alsoinclude a light source configured to direct light to an assay region ofthe flow channel. The system may include a detector configured toreceive a signal from an assay region of the flow channel, wherein thesignal is provided by the fluorescent composition. Optionally further,the sample analysis system may include one or more additional detectorsand/or light sources for the detection of one or more additionalsignals.

In certain aspects, the system may further include computer-basedsystems configured to detect the presence of the fluorescent signal. A“computer-based system” refers to the hardware means, software means,and data storage means used to analyze the information of the presentinvention. The minimum hardware of the computer-based systems of thepresent invention includes a central processing unit (CPU), input means,output means, and data storage means. A skilled artisan can readilyappreciate that any one of the currently available computer-based systemare suitable for use in the present invention. The data storage meansmay include any manufacture including a recording of the presentinformation as described above, or a memory access means that can accesssuch a manufacture.

To “record” data, programming or other information on a computerreadable medium refers to a process for storing information, using anysuch methods as known in the art. Any convenient data storage structuremay be chosen, based on the means used to access the stored information.A variety of data processor programs and formats can be used forstorage, e.g., word processing text file, database format, etc.

A “processor” references any hardware and/or software combination thatwill perform the functions required of it. For example, any processorherein may be a programmable digital microprocessor such as available inthe form of an electronic controller, mainframe, server or personalcomputer (desktop or portable). Where the processor is programmable,suitable programming can be communicated from a remote location to theprocessor, or previously saved in a computer program product (such as aportable or fixed computer readable storage medium, whether magnetic,optical or solid state device based). For example, a magnetic medium oroptical disk may carry the programming, and can be read by a suitablereader communicating with each processor at its corresponding station.

In addition to the sensor device and signal processing module, e.g., asdescribed above, systems of the invention may include a number ofadditional components, such as data output devices, e.g., monitorsand/or speakers, data input devices, e.g., interface ports, keyboards,etc., fluid handling components, power sources, etc.

In certain aspects, the system includes a flow cytometer. Flowcytometers of interest include, but are not limited, to those devicesdescribed in U.S. Pat. Nos. 4,704,891; 4,727,029; 4,745,285; 4,867,908;5,342,790; 5,620,842; 5,627,037; 5,701,012; 5,895,922; and 6,287,791;the disclosures of which are herein incorporated by reference.

Other systems may find use in practicing the subject methods. In certainaspects, the system may be a fluorimeter or microscope loaded with asample having a fluorescent composition of any of the embodimentsdiscussed herein. The fluorimeter or microscope may include a lightsource configured to direct light to the assay region of the flowchannel. The fluorimeter or microscope may also include a detectorconfigured to receive a signal from an assay region of the flow channel,wherein the signal is provided by the fluorescent composition.

Utility

The chromophore compositions as described herein may find use in avariety of methodologies in which labeling of a sample is desirable.Such methodologies include but are not limited to cytometry, microscopy,immunoassays (e.g. competitive or non-competitive), assessment of a freeanalyte, assessment of receptor bound ligand, and so forth. Thecompositions described herein may be useful in analysis of any of anumber of samples, including but not limited to biological fluids, cellculture samples, and tissue samples. In certain aspects, the chromophorecompositions described herein may find use in methods where analytes aredetected in a sample using fluorescent labels, such as in fluorescentactivated cell sorting or analysis, immunoassays, immunostaining, andthe like. FIG. 7 demonstrates that fluorescent compositions of certainembodiments disclosed herein are stable and may be used in peripheralblood samples including free hemin (e.g., the fluorescent dye motif maynot be outcompeted by free hemin for the prosthetic group binding cavityof the metalloprotein).

The subject chromophore compositions may find use in enhancing theproperties of certain environmentally sensitive dyes. Such dyes mayexhibit undesirable fluorescence properties (e.g., excitation oremission spectrum, fluorescence lifetime, quantum yield, etc.), lowsolubility, or other characteristics in specific environments, such asaqueous environments, organic environments, certain pH, and certaintemperatures. In some aspects, environmentally sensitive fluorescentdyes used to label antibodies used in analysis and sorting of bloodcells may exhibit poor fluorescence of these molecules in an aqueousenvironment. Some environmentally sensitive dyes fluoresce only in anarrow range of environmental conditions. The methods and compositionsdiscussed herein provide, among other benefits, labeling reagents thatare sensitive, and easily detected, in a wider range of environmentalconditions.

In addition, protein dye encapsulation systems of fluorescent moleculessuch as GFP, PE, APC or PerCP, may require the fluorophore be covalentlylinked to the protein moiety. The fluorescent composition and methods ofthe subject invention provide fluorescent proteins without covalentattachment of the fluorescent dye moiety. This beneficially provides foran environmentally sensitive fluorescent molecule bound in an organicenvironment that is soluble in an aqueous environment. The boundfluorescent molecule may not be covalently attached to the heme protein,insuring that the fluorescence properties of the molecule are notnegatively impacted.

In certain embodiments of the invention, apo-Mb is a generic dyeencapsulation system that allows one to define and amplify dyes bydesign. The light amplification system is achieved using standardcross-linking reagents. As compared to PE fluorophores, a six to tenfold increase in signal relative to size and weight may be achieved.

Embodiments wherein an environmentally sensitive dye is complexed with aheme-protein may be used in combination with an antigen-specific reagent(also referred to as a specific binding domain), and provides for theuse of such dyes in a number of biological detection assays.

Kits

Aspects of the invention further include kits for use in practicing thesubject methods and compositions. The compositions of the invention canbe included as reagents in kits either as starting materials (e.g., suchas chromophore-bound heme-protein including a bi-functional linkermolecule) or pre-formed complexes (e.g., such as a chromophore-boundheme-protein conjugated to antigen specific antibody) provided for usein, for example, the methodologies described above.

A kit may include the fluorescent composition of any of the embodimentsof the subject compositions. The kit may further include a container(e.g., tube, bottle, packet, etc.) for providing the fluorescentcomposition. In certain aspects, the fluorescent composition provided bythe kit may include a linker conjugated to a metalloprotein of thefluorescent composition. The linker may be functionalized (e.g., tocovalently attach to an antibody or other specific binding domain).

In certain aspects, the kit may also include one or more additionalfluorescent compositions. The one or more additional fluorescentcompositions may be provided in separate containers (e.g., separatetubes, bottles, or wells in a multi-well strip or plate).

In certain aspects, the kit may further include reagents for performinga flow cytometric assay. Examples of said reagents include buffers forat least one of reconstitution and dilution of the first and seconddetectible molecules, buffers for contacting a cell sample with one orboth of the first and second detectible molecules, wash buffers, controlcells, control beads, fluorescent beads for flow cytometer calibrationand combinations thereof. The kit may also include one or more cellfixing reagents such as paraformaldehyde, glutaraldehyde, methanol,acetone, formalin, or any combinations or buffers thereof. Further, thekit may include a cell permeabilizing reagent, such as methanol, acetoneor a detergent (e.g., triton, NP-40, saponin, tween 20, digitonin,leucoperm, or any combinations or buffers thereof. Other proteintransport inhibitors, cell fixing reagents and cell permeabilizingreagents familiar to the skilled artisan are within the scope of thesubject kits.

The fluorescent composition may be provided in a liquid composition,such as any suitable buffer. Alternatively, the fluorescent compositionmay be provided in a dry composition (e.g., may be lyophilized), and thekit may optionally include one or more buffers for reconstituting thedry composition. In certain aspects, the kit may include aliquots of thefluorescent composition provided in separate containers (e.g., separatetubes, bottles, or wells in a multi-well strip or plate).

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, DVD, portable flash drive, etc., on which theinformation has been recorded. Yet another means that may be present isa website address which may be used via the internet to access theinformation at a removed site. Any convenient means may be present inthe kits.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

The following experiments demonstrate that when an environmentallysensitive dye is encapsulated in a heme-protein, the fluorescence of thedye is increased in an aqueous solution.

Apo-myoglobin (Apo-Mb) was examined as an encapsulation system for waterinsoluble and/or environmentally-sensitive fluorescent organic dyes. Theutility of Apo-Mb was studied with and without the heme-coordinatinghistidine residues intact (e.g., H64 and H93 of human myoglobin, as seenin the molecular structure shown in FIG. 1). The protein was alteredusing recombinant techniques to maximize dye interactions whiledecreasing its affinity for the “heme” structure that naturally occursin myoglobin. Histidine amino acid replacement included alanine,leucine, phenylalanine and tryptophan. In addition, amino acid residuesabove and below the edge of the heme-binding site were replaced withcysteines. These recombinant modifications were made for collapsing thecavity with a cysteine reactive bis-maleimide crosslinker post dyedocking in the protein cavity. An alternate method for securing the dyewas fixation using other standard chemical methods. Apo-Mb basedencapsulation creates new fluorescent proteins without covalentattachment of a fluorophore. Post dye incorporation, the protein/dyemonomer was externally cross-linked with 6 to 10 monomers creating afluorescence enhancing system for the monomeric dye.

The technical principle is to generate a generic protein encapsulationsystem utilizing the naturally occurring cavity in the myoglobin(FIG. 1) that is capable of associating with the “heme” structure inliving organisms (FIG. 2). Replacing the heme with an environmentallysensitive dye such those in the squaraine family (FIG. 3) and collapsingthe cavity generates a stable monomer that fluoresces in aqueous media(FIG. 4). The nature of the parent protein affords a non-polarenvironment and/or solubility to the organic dye. Once docked the cavitymay be collapsed via sulfhydryl-maleimide chemistry and/or fixation viaparaformaldehyde.

Expression and Purification of Apo-Mb and its Mutants

Human myoglobin was cloned from IMAGE clone 4244838 into the pVEXHNexpression vector via HindIII/EcoRI restriction sites. Proteinexpression was induced in the BL21 strain of E. coli by adding 0.5 mMIPTG to a bacterial culture grown in M9 minimal medium to an OD600 of0.8. After overnight incubation at room temperature the bacteria werepelleted and resuspended in 150 mM NaCl, 1 mM mercaptoethanol, 25 mM Naphosphate buffer, pH 7.4. Cells were sonified followed by 30 mincentrifugation at 3000 g. Apo-Mb was purified on Ni-NTA agarose (QIAGEN)and eluted with 250 mM imidazole. The protein was further purified bysize exclusion chromatography on Superdex 75 10/300 or HiLoad Superdex7516/60 column in 150 mM NaCV25 mM Na phosphate buffer, pH 7.4. Theresulting Apo-Mb is free from heme group and shows lack of lightabsorption at 409 nm, as seen if FIG. 2.

Example 1

In Example 1, an encapsulation system for water insoluble and/orenvironmentally-sensitive fluorescent organic dyes was examined. Thestability of such complexes was studied with and without the hemecoordinating histidine residues of the protein intact. The protein wasaltered using recombinant techniques to maximize dye interactions whiledecreasing its affinity for the “heme” structure that naturally occursin myoglobin. Histidine amino acid replacement included alanine,leucine, phenylalanine and tryptophan. In addition, amino acid residuesabove and below the edge of the heme binding site were replaced withcysteines. These recombinant modifications were made for locking thecavity with a cysteine reactive bis-maleimide (BS-3) crosslinker afterbinding the fluorescent organic molecule in the protein cavity. Thebinding affinity of the fluorescent molecule was measured againstcompetition from a hemin molecule.

FIG. 7 shows the results of one such binding assay. Human recombinantapo-Mb was incubated with 2× excess of mono-squaraine in 50 mM sodiumphosphate buffer, 150 mM sodium chloride, pH 7.2. Then, 0.5 ml samplesof the apo-Mb/mono-squaraine complex containing 25 nmoles of apo-Mb wereincubated for 2 hours with 175, 250 and 375 nmols of BS3 reagent (7, 10and 15× crosslinker excess, respectively). The fluorescent signal ofmono-squaraine-apo-myoglobin complex was measured as a function ofincreasing hemin concentration. They dye-protein complex was treatedwith three different concentration of crosslinking reagent bismaleimide(BS-3). It can be seen from the graph of FIG. 4 that crosslinkedapo-myoglobin is significantly more resistant to displacement of thefluorophore by hemin than non-crosslinked apo-myoglobin. Thisbeneficially provides for a stable fluorescent reagent that may be usedin peripheral blood samples including free hemin.

Example 2

The His64 and His93 amino acids of apo-myoglobin (apo-Mb) weregenetically modified to form a modified apo-Mb with an improved affinityfor mono- or bis-squaraines. Histidine amino acid replacement includedalanine, leucine, phenylalanine and tryptophan. Shown below in Table 1are binding constants for a variety of genetically modified apo-MBspecies, indicating that the affinity of mono- and bis-squaraine toapo-Mb may be modified by genetic modification of histidine residues inthe protein.

TABLE 1 MonoSQ BisSQ MonoSQ % from Kd BisSQ % from Kd Kd (nM) ApoMb Kd(nM) ApoMb ApoMb 2.3 100 13.3 100 (H64/H93) A64 3.1 134.8 2 15 L64 2.191.3 11.5 86.5 F64 4.8 208.7 15.5 116.5 W64 6.2 269.6 34.8 261.7 A93 5.1221.7 3.7 27.8 L93 4.9 213 15.2 114.3 F93 2.1 91.3 23.1 173.7 W93 4.5195.7 8.4 63.2

Through amino acid replacement the dye binding is optimized (FIG. 8).Amino acid SEQ ID NO:01 is shown in FIG. 8. Amino acids 3 to 14 of SEQID NO:01 are of a histidine tag enabling purification by Ni-NTAaffinity. M27 of SEQ ID NO:01 corresponds to the initiator methionine ofthe wild-type human myoglobin sequence. Amino acids 28 to 180 correspondto a human myoglobin sequence of 153 amino acids with the followingmutations: H64 of the wild-type sequence has been replaced with alanine(A91 of SEQ ID NO:01); H93 of the wild-type sequence has been replacedwith phenylalanine (F120 of SEQ ID NO:01); and amino acid residues aboveand below the edge of the heme-binding site were replaced with cysteines(C94 and C119 of SEQ ID NO:01). In certain aspects, the amino acidsequence starting with M27 or G28 of SEQ ID NO:01 may be suitable forany embodiments of the invention described herein.

Notwithstanding the appended clauses, the disclosure is also defined bythe following clauses:

1. A fluorescent composition, the composition comprising:

a fluorescent component comprising:

-   -   (i) a metalloprotein comprising a prosthetic group binding        cavity; and    -   (ii) a fluorescent dye moiety stably associated with the        prosthetic group binding cavity;

wherein the metalloprotein comprises a stabilizing modification thatenhances association of the dye moiety with the cavity.

2. The composition according to Clause 1, wherein the prosthetic groupbinding cavity has a volume ranging from 50 nm³ to 1000 nm³.3. The composition according to Clause 2, wherein the metalloprotein isa heme binding protein or mutant thereof.4. The composition according to Clause 3, wherein the heme bindingprotein is an apo-myoglobin.5. The composition according to Clause 4, wherein the apo-myoglobin hasa wild-type sequence.6. The composition according to Clause 4, wherein the apo-myoglobinincludes one or more mutations.7. The composition according to Clause 6, wherein the mutation is apoint mutation.8. The composition according to Clause 7, wherein the point mutationcomprises a substitution of a cysteine residue for a naturally occurringamino acid residue.9. The composition according to Clause 7, wherein the point mutationcomprises a substitution of a non-histidine residue for a naturallyoccurring histidine residue.10. The composition according to Clause 9, wherein the non-histidineresidue is selected from the group consisting of alanine, leucine,phenylalanine, and tryptophan.11. The composition according to any of the preceding clauses, whereinthe fluorescent dye moiety comprises a non-protein organic fluorophore.12. The composition according to Clause 11, wherein the organicfluorophore has a molecular weight ranging from 50 Da to 5 kDa.13. The composition according to Clause 11, wherein the organicfluorophore is an environmentally sensitive fluorophore.14. The composition according to Clause 13, wherein an environmentallysensitive fluorophore stably associated with the prosthetic groupbinding cavity of the metalloprotein has a first fluorescence intensitythat is higher than a second fluorescence intensity of theenvironmentally sensitive fluorophore free in aqueous solution.15. The method of Clause 14, wherein the first fluorescence intensity isat least 50 times higher than the second fluorescence intensity.16. The composition according to Clause 13, wherein the environmentallysensitive fluorophore is a squaraine dye.17. The composition according to any of the preceding clauses, whereinthe fluorescent dye moiety is non-covalently bound to the prostheticbinding group cavity.18. The composition according to any of the preceding clauses, whereinthe composition further comprises a specific binding domain.19. The composition according to Clause 18, wherein the specific bindingdomain has an affinity for an analyte of K_(A) of 10⁴ M⁻¹ or greater.20. The composition according to Clause 19, wherein the specific bindingdomain comprises a peptidic or polypeptidic moiety.21. The composition according to Clause 20, wherein the peptidic orpolypeptidic moiety comprises an antibody or binding fragment thereof.22. The composition according to Clause 21, wherein the peptidic orpolypeptidic moiety specifically binds a cell surface marker.23. The composition according to Clause 18, wherein the specific bindingdomain is a ligand that specifically binds a cell surface receptor.24. The composition according to Clause 18, wherein the specific bindingdomain comprises a nucleic acid moiety.25. The composition according to any of the preceding clauses, furthercomprising a linker conjugated to the metalloprotein.26. The composition according to any of the preceding clauses, whereinthe composition comprises two or more fluorescent components covalentlybound to each other.27. The composition according to any of the preceding clauses, whereinthe stabilizing modification comprises an internal crosslinking of themetalloprotein.28. The composition according to Clause 27, wherein the internalcrosslinking comprises crosslinked cysteine residues.29. The composition according to Clause 27, wherein the internalcrosslinking comprises crosslinked lysine residues.30. A fluorescent composition, the composition comprising:

a fluorescent component comprising:

-   -   (i) a metalloprotein comprising a prosthetic group binding        cavity; and    -   (ii) an environmentally sensitive fluorescent dye moiety stably        associated with the prosthetic group binding cavity.        31. A method of assaying a sample, the method comprising:

contacting the sample with a fluorescent composition, wherein thefluorescent composition comprises:

-   -   i) a metalloprotein comprising a prosthetic group binding        cavity;    -   ii) a fluorescent dye moiety stably associated with the        prosthetic group binding cavity; and

wherein the metalloprotein comprises a stabilizing modification thatenhances association of the dye moiety with the cavity.

32. The method according to Clause 31, wherein the fluorescentcomposition further comprises a specific binding domain.33. The method according to Clause 32, further comprising obtaining afluorescence signal from the sample.34. The method according to Clause 33, further comprising evaluating ananalyte based on the fluorescence signal.35. The method according to Clause 34, wherein evaluating comprisesassessing the amount of the analyte in the sample.36. The method according to Clause 33, further comprising assessing theamount of a ligand as an inverse function of the intensity of thefluorescence signal, wherein the ligand competes for the analyte boundby the specific binding domain.37. The method according to any of Clauses 31 to 36, wherein thebiological sample is a cell sample.38. The method according to any of Clauses 31 to 37, wherein theprosthetic group binding cavity has a volume ranging from 50 nm³ to 1000nm³.39. The method according to any of Clauses 31 to 38, wherein themetalloprotein is a heme binding protein or mutant thereof.40. The method according to Clause 39, wherein the heme binding proteinis an apo-myoglobin.41. The method according to Clause 40, wherein the apo-myoglobin has awild-type sequence.42. The method according to Clause 40, wherein the apo-myoglobinincludes one or more mutations.43. The method according to Clause 42, wherein the mutation is a pointmutation.44. The method according to Clause 43, wherein the point mutationcomprises a substitution of a cysteine for a naturally occurring aminoacid residue.45. The method according to Clause 44, wherein the point mutationcomprises a substitution of a non-histidine residue for a naturallyoccurring histidine residue.46. The method according to Clause 45, wherein the non-histidine residueis selected from the group consisting of alanine, leucine,phenylalanine, and tryptophan.47. The method according to any of Clauses 31 to 46, wherein thefluorescent dye moiety comprises a non-protein organic fluorophore.48. The method according to Clause 47, wherein the organic fluorophorehas a molecular weight ranging from 50 Da to 5 kDa.49. The method according to Clause 48, wherein the organic fluorophoreis an environmentally sensitive fluorophore.50. The method according to Clause 49, wherein an environmentallysensitive fluorophore stably associated with the prosthetic groupbinding cavity of the metalloprotein has a first fluorescence intensitythat is higher than a second fluorescence intensity of theenvironmentally sensitive fluorophore free in aqueous solution.51. The method according to Clause 50, wherein the first fluorescenceintensity is at least 50 times higher than the second fluorescenceintensity.52. The method according to Clause 49 or 50, wherein the environmentallysensitive fluorophore is a squaraine dye.53. The method according to any of Clauses 31 to 52, wherein thefluorescent dye moiety is non-covalently bound to the prosthetic bindinggroup cavity.54. The method according to any of Clauses 32 to 53, wherein thespecific binding domain has an affinity for an analyte of K_(A) of 10⁴M⁻¹ or greater.55. The method according to Clause 54, wherein the specific bindingdomain comprises a peptidic or polypeptidic moiety.56. The method according to Clause 55, wherein the peptidic orpolypeptidic moiety comprises an antibody or binding fragment thereof.57. The method according to Clause 56, wherein the peptidic orpolypeptidic moiety specifically binds a cell surface marker.58. The method according to any of Clauses 32 to 53, wherein thespecific binding domain is a ligand that specifically binds a cellsurface receptor.59. The method according to any of Clauses 32 to 53, wherein thespecific binding domain comprises a nucleic acid moiety.60. The method according to any of Clauses 31 59, wherein thefluorescent composition comprises two or more fluorescent compositionscovalently bound to each other.61. The method according to any of Clauses 31 to 60, wherein thestabilizing modification comprises an internal crosslinking of themetalloprotein.62. The method according to Clause 61, wherein the internal crosslinkingcomprises crosslinking cysteine residues.63. The method according to Clauses 61 or 62, wherein the internalcrosslinking comprises crosslinking lysine residues.64. A method of producing a fluorescent composition, the methodcomprising:

contacting a metalloprotein having an empty prosthetic group bindingcavity with a fluorescent dye moiety in a manner sufficient for thefluorescent dye moiety to enter the prosthetic group binding cavity, and

stably associating the fluorescent dye moiety with the prosthetic groupbinding cavity.

65. The method according to Clause 64, wherein stably associating thefluorescent dye moiety with the prosthetic group binding cavitycomprises modifying the metalloprotein.66. The method according to Clause 65, wherein modifying themetalloprotein comprises generating an internal crosslink in themetalloprotein after the dye enters the prosthetic group binding cavity.67. The method according to Clause 66, wherein a cysteine residue of themetalloprotein replaces a non-cysteine residue of a naturally occurringmetalloprotein.68. The method according to Clause 66, further comprising modulating pHprior to generating an internal crosslink in the metalloprotein.69. The method according to any of Clauses 64 to 68, wherein theprosthetic group binding cavity has a volume ranging from 50 nm³ to 1000nm³.70. The method according to any of Clauses 64 to 69, wherein themetalloprotein is a heme binding protein or mutant thereof.71. The method according to Clause 70, wherein the heme binding proteinis an apo-myoglobin.72. The method according to Clause 71, wherein the apo-myoglobin has awild-type sequence.73. The method according to Clause 71, wherein the apo-myoglobinincludes one or more mutations.74. The method according to Clause 73, wherein the mutation is a pointmutation.75. The method according to Clause 74, wherein the point mutationcomprises a substitution of a cysteine for a naturally occurring aminoacid residue.76. The method according to Clause 74, wherein the point mutationcomprises a substitution of a non-histidine residue for a naturallyoccurring histidine residue.77. The method according to Clause 76, wherein the non-histidine residueis selected from the group consisting of alanine, leucine,phenylalanine, and tryptophan.78. The method according to any of Clauses 64 to 77, wherein thefluorescent dye moiety comprises a non-protein organic fluorophore.79. The method according to Clause 78, wherein the organic fluorophorehas a molecular weight ranging from 50 Da to 5 kDa.80. The method according to Clause 79, wherein the organic fluorophoreis an environmentally sensitive fluorophore.81. The method according to Clause 80, wherein an environmentallysensitive fluorophore stably associated with the prosthetic groupbinding cavity of the metalloprotein has a first fluorescence intensitythat is higher than a second fluorescence intensity of theenvironmentally sensitive fluorophore free in aqueous solution.82. The method according to Clause 81, wherein the first fluorescenceintensity is at least 50 times higher than the second fluorescenceintensity.83. The method according to Clauses 80, 81 or 82, wherein theenvironmentally sensitive fluorophore is a squaraine dye.84. The method according to any of Clauses 64 to 83, wherein thefluorescent dye moiety is non-covalently bound to the prosthetic bindinggroup cavity.85. The method according to any of Clauses 64 to 84, wherein thefluorescent composition further comprises a specific binding domain.86. The method according to Clause 85, wherein the specific bindingdomain has an affinity for an analyte of K_(A) of 10⁴ M⁻¹ or greater.87. The method according to Clause 86, wherein the specific bindingdomain comprises a peptidic or polypeptidic moiety.88. The method according to Clause 87, wherein the peptidic orpolypeptidic moiety comprises an antibody or binding fragment thereof.89. The method according to Clause 88, wherein the peptidic orpolypeptidic moiety specifically binds a cell surface marker.90. The method according to Clause 85, wherein the specific bindingdomain is a ligand that specifically binds a cell surface receptor.91. The method according to Clause 85, wherein the specific bindingdomain comprises a nucleic acid moiety.92. The method according to any of Clauses 64 to 91, wherein thefluorescent composition comprises two or more fluorescent compositionscovalently bound to each other.93. A method of producing a metalloprotein suitable for binding to afluorescent dye moiety, the method comprising:

expressing a mutant metalloprotein in a host cell, wherein the mutantmetalloprotein comprises a mutation in a prosthetic group bindingcavity; and

harvesting the mutant metalloprotein from the host cell.

94. The method according to Clause 93, wherein the prosthetic groupbinding cavity has a volume ranging from 50 nm³ to 1000 nm³.95. The method according to Clause 94, wherein the metalloprotein is aheme binding protein.96. The method according to Clause 95, wherein the heme binding proteinis an apo-myoglobin.97. The method according to Clause 96, wherein the apo-myoglobinincludes one or more mutations.98. The method according to Clause 97, wherein the mutations comprise atleast one of a substitution, point mutation, insertion, and a deletion.99. The method according to Clause 97, wherein the mutation is a pointmutation.100. The method according to Clause 99, wherein the point mutationcomprises a substitution of a cysteine for a naturally occurring aminoacid residue.101. The method according to Clause 99, wherein the point mutationcomprises a substitution of a non-histidine residue for a naturallyoccurring histidine residue.102. The method according to Clause 101, wherein the non-histidineresidue is selected from the group consisting of alanine, leucine,phenylalanine, and tryptophan.103. The method according to any of Clauses 93 to 102, wherein the hostcell is of a bacterial strain.104. The method according to any of Clauses 93 to 103, wherein the hostcell is an animal cell.105. The method according to Clause 104, wherein the animal cell is of ayeast strain.106. The method according to any of Clauses 93 to 105, whereinexpressing comprises exposing the host cell to a vector comprising anucleotide sequence encoding the mutant metalloprotein.107. The method according to claim 106, wherein the method furthercomprises maintaining the exposed host cell under conditions sufficientfor the nucleotide sequence to be expressed.108. The method according to Clauses 106 or 107, wherein the vector is avirus.109. The method according to Clauses 106 or 107, wherein the vector is aplasmid.110. The method according to any of Clauses 106 to 109, wherein thenucleotide sequence is under the control of a promoter.111. The method according to Clause 110, wherein the expression isinducible.112. The method according to Clause 111, further comprising exposing thehost cell to an inducing agent.113. A kit comprising:

a fluorescent composition, the fluorescent composition comprising:

-   -   (i) a metalloprotein comprising a prosthetic group binding        cavity; and    -   (ii) a fluorescent dye moiety stably associated with the        prosthetic group binding cavity;

wherein the metalloprotein comprises a stabilizing modification thatenhances association of the dye moiety with the cavity; and

a container comprising the fluorescent composition.

114. The kit according to Clause 113, wherein the stabilizingmodification comprises an internal crosslinking of the metalloprotein.115. The kit according to Clause 114, wherein the internal crosslinkingcomprises crosslinked cysteine residues.116. The kit according to Clause 114, wherein the internal crosslinkingcomprises crosslinked lysine residues.117. The kit according to any of Clauses 113 to 116, wherein theprosthetic group binding cavity has a volume ranging from 50 nm³ to 1000nm³.118. The kit according to any of Clauses 113 to 117, wherein themetalloprotein is a heme binding protein or mutant thereof.119. The kit according to Clause 118, wherein the heme binding proteinis an apo-myoglobin.120. The kit according to Clause 119, wherein the apo-myoglobin has awild-type sequence.121. The kit according to Clause 120, wherein the apo-myoglobin includesone or more mutations.122. The kit according to Clause 121, wherein the mutation is a pointmutation.123. The kit according to Clause 122, wherein the point mutationcomprises a substitution of a cysteine for a naturally occurring aminoacid residue.124. The kit according to Clause 122, wherein the point mutationcomprises a substitution of a non-histidine residue for a naturallyoccurring histidine residue.125. The kit according to Clause 124, wherein the non-histidine residueis selected from the group consisting of alanine, leucine,phenylalanine, and tryptophan.126. The kit according to any of Clauses 113 to 125, wherein thefluorescent dye moiety comprises a non-protein organic fluorophore.127. The kit according to Clause 126, wherein the organic fluorophorehas a molecular weight ranging from 50 Da to 5 kDa.128. The kit according to Clause 127, wherein the organic fluorophore isan environmentally sensitive fluorophore.129. The kit according to Clause 128, wherein an environmentallysensitive fluorophore stably associated with the prosthetic groupbinding cavity of the metalloprotein has a first fluorescence intensitythat is higher than a second fluorescence intensity of theenvironmentally sensitive fluorophore free in aqueous solution.130. The kit according to Clause 129, wherein the first fluorescenceintensity is at least 50 times higher than the second fluorescenceintensity.131. The kit according to Clause 128, wherein the environmentallysensitive fluorophore is a squaraine dye.132. The kit according to any of Clauses 113 to 131, wherein thefluorescent dye moiety is non-covalently bound to the prosthetic bindinggroup cavity.133. The kit according to any of Clauses 113 to 132, wherein thefluorescent composition further comprises a specific binding domain.134. The kit according to Clause 133, wherein the specific bindingdomain has an affinity for an analyte of K_(A) of 10⁴ M⁻¹ or greater.135. The kit according to Clause 134, wherein the specific bindingdomain comprises a peptidic or polypeptidic moiety.136. The kit according to Clause 135, wherein the peptidic orpolypeptidic moiety comprises an antibody or binding fragment thereof.137. The kit according to Clause 136, wherein the peptidic orpolypeptidic moiety specifically binds a cell surface marker.138. The kit according to Clause 133, wherein the specific bindingdomain is a ligand that specifically binds a cell surface receptor.139. The kit according to Clause 133, wherein the specific bindingdomain comprises a nucleic acid moiety.140. The kit according to any of Clauses 113 to 139, wherein thefluorescent composition further comprises a linker conjugated to themetalloprotein.141. The kit according to any of Clauses 113 to 140, wherein thefluorescent composition comprises two or more fluorescent compositionscovalently bound to each other.142. The kit according to any of Clauses 113 to 141, further comprisingreagents for performing a flow cytometric assay.143. The kit according to any of Clauses 113 to 142, further comprisingone or more additional fluorescent compositions.144. The kit according to any of Clauses 113 to 143, wherein thefluorescent composition is in a liquid composition.145. The kit according to any of Clauses 113 to 143, wherein thefluorescent composition is in a dry composition.146. A sample analysis system comprising:

a flow channel loaded with a sample comprising a fluorescentcomposition;

wherein the fluorescent composition comprises:

-   -   (i) a metalloprotein comprising a prosthetic group binding        cavity; and    -   (ii) a fluorescent dye moiety stably associated with the        prosthetic group binding cavity.        147. The system according to Clause 146, further comprising a        light source configured to direct light to an assay region of        the flow channel.        148. The system according to Clause 147, further comprising a        detector configured to receive a signal from an assay region of        the flow channel, wherein the signal is provided by the        fluorescent composition.        149. The system according to any of Clauses 146 to 148, wherein        the metalloprotein comprises a stabilizing modification that        enhances association of the dye moiety with the cavity.        150. The system according to Clause 149, wherein the stabilizing        modification comprises an internal crosslinking of the        metalloprotein.        151. The system according to Clause 150, wherein the internal        crosslinking comprises crosslinked cysteine residues.        152. The system according to Clause 151, wherein the internal        crosslinking comprises crosslinked lysine residues.        153. The system according to any of Clauses 146 to 152, wherein        the prosthetic group binding cavity has a volume ranging from 50        nm³ to 1000 nm³.        154. The system according to any of Clauses 146 to 153, wherein        the metalloprotein is a heme binding protein or mutant thereof.        155. The system according to Clause 154, wherein the heme        binding protein is an apo-myoglobin.        156. The system according to Clause 155, wherein the        apo-myoglobin has a wild-type sequence.        157. The system according to Clause 155, wherein the        apo-myoglobin includes one or more mutations.        158. The system according to Clause 157, wherein the mutation is        a point mutation.        159. The system according to Clause 158, wherein the point        mutation comprises a substitution of a cysteine for a naturally        occurring amino acid residue.        160. The system according to Clause 158, wherein the point        mutation comprises a substitution of a non-histidine residue for        a naturally occurring histidine residue.        161. The system according to Clause 160, wherein the        non-histidine residue is selected from the group consisting of        alanine, leucine, phenylalanine, and tryptophan.        162. The system according to any of Clauses 146 to 161, wherein        the fluorescent dye moiety comprises a non-protein organic        fluorophore.        163. The system according to Clause 162, wherein the organic        fluorophore has a molecular weight ranging from 50 Da to 5 kDa.        164. The system according to Clause 163, wherein the organic        fluorophore is an environmentally sensitive fluorophore.        165. The system according to Clause 164, wherein an        environmentally sensitive fluorophore stably associated with the        prosthetic group binding cavity of the metalloprotein has a        first fluorescence intensity that is higher than a second        fluorescence intensity of the environmentally sensitive        fluorophore free in aqueous solution.        166. The system according to Clause 165, wherein the first        fluorescence intensity is at least 50 times higher than the        second fluorescence intensity.        167. The system according to Clause 164, wherein the        environmentally sensitive fluorophore is a squaraine dye.        168. The system according to any of Clauses 146 to 167, wherein        the fluorescent dye moiety is non-covalently bound to the        prosthetic binding group cavity.        169. The system according to any of Clause 146 to 168, wherein        the fluorescent composition further comprises a specific binding        domain.        170. The system according to Clause 169, wherein the specific        binding domain has an affinity for an analyte of K_(A) of 10⁴        M⁻¹ or greater.        171. The system according to Clause 169, wherein the specific        binding domain comprises a peptidic or polypeptidic moiety.        172. The system according to Clause 171, wherein the peptidic or        polypeptidic moiety comprises an antibody or binding fragment        thereof.        173. The system according to Clause 172, wherein the peptidic or        polypeptidic moiety specifically binds a cell surface marker.        174. The system according to Clause 169, wherein the specific        binding domain is a ligand that specifically binds a cell        surface receptor.        175. The system according to Clause 169, wherein the specific        binding domain comprises a nucleic acid moiety.        176. The system according to any of Clauses 146 to 175, wherein        the fluorescent composition comprises two or more fluorescent        compositions covalently bound to each other.        177. The system according to any of Clauses 146 to 176, wherein        the sample comprises one or more additional fluorescent        compositions.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A fluorescent composition, the compositioncomprising: a fluorescent component comprising: (i) a metalloproteincomprising a prosthetic group binding cavity; and (ii) a fluorescent dyemoiety stably associated with the prosthetic group binding cavity;wherein the metalloprotein comprises a stabilizing modification thatenhances association of the dye moiety with the cavity.
 2. Thecomposition according to claim 1, wherein the prosthetic group bindingcavity has a volume ranging from 50 nm³ to 1000 nm³.
 3. The compositionaccording to claim 2, wherein the metalloprotein is a heme bindingprotein or mutant thereof.
 4. The composition according to claim 3,wherein the heme binding protein is an apo-myoglobin.
 5. The compositionaccording to claim 4, wherein the apo-myoglobin has a wild-typesequence.
 6. The composition according to claim 4, wherein theapo-myoglobin includes one or more mutations.
 7. The compositionaccording to claim 6, wherein the mutation is a point mutation.
 8. Thecomposition according to claim 1, wherein the fluorescent dye moietycomprises a non-protein organic fluorophore.
 9. The compositionaccording to claim 8, wherein the non-protein organic fluorophore is anenvironmentally sensitive fluorophore.
 10. The composition according toclaim 9, wherein the environmentally sensitive fluorophore stablyassociated with the prosthetic group binding cavity of themetalloprotein has a first fluorescence intensity that is higher than asecond fluorescence intensity of the environmentally sensitivefluorophore free in aqueous solution.
 11. The method according to claim10, wherein the first fluorescence intensity is at least 50 times higherthan the second fluorescence intensity.
 12. The composition according toclaim 9, wherein the environmentally sensitive fluorophore is asquaraine dye.
 13. The composition according to claim 1, wherein thefluorescent dye moiety is non-covalently bound to the prosthetic bindinggroup cavity.
 14. The composition according to claim 1, wherein thecomposition further comprises a specific binding domain.
 15. Thecomposition according to claim 1, further comprising a linker conjugatedto the metalloprotein.
 16. The composition according to claim 1, whereinthe composition comprises two or more fluorescent components covalentlybound to each other.
 17. The composition according to claim 1, whereinthe stabilizing modification comprises an internal crosslinking of themetalloprotein.
 18. A fluorescent composition, the compositioncomprising: a fluorescent component comprising: (i) a metalloproteincomprising a prosthetic group binding cavity; and (ii) anenvironmentally sensitive fluorescent dye moiety stably associated withthe prosthetic group binding cavity.
 19. A method of assaying a sample,the method comprising: contacting the sample with a fluorescentcomposition, wherein the fluorescent composition comprises: i) ametalloprotein comprising a prosthetic group binding cavity; ii) afluorescent dye moiety stably associated with the prosthetic groupbinding cavity.
 20. A kit comprising: a fluorescent composition, thefluorescent composition comprising: (i) a metalloprotein comprising aprosthetic group binding cavity; and (ii) a fluorescent dye moietystably associated with the prosthetic group binding cavity; and acontainer comprising the fluorescent composition.